Nearly 400 million people worldwide are chronically infected with hepatitis B virus (HBV) and are at risk for cirrhosis or liver cancer, which necessitates liver transplantation. Immunization with HBV envelope proteins prevents HBV infection, and hepatitis B immune globulin (HBIG), alone or together with nucleoside analogs such as lamivudine, reduces the re- infection rate of the grafted liver. However, breakthrough infection can occur in both vaccine recipients and liver graft recipients due to the emergence of neutralization escape mutants, often together with small amounts of wild-type virus. Such mutants contain amino acid changes in the alpha determinant of viral envelope proteins, the primary target of neutralizing antibodies. Our preliminary studies suggest that structural changes around the alpha determinant may impair virion secretion, but the possibility of the co-infecting wild-type virus or second-site mutations restoring virion secretion cannot be excluded. In the present study, we propose to analyze the virion secretion efficiencies of 21 alpha determinant mutants and 7 mutants associated with drug resistance. For mutants with defective or nearly defective virion secretion, which are nevertheless detectable in patient blood, we will determine whether the defect can be overcome by a small amount of wild-type envelope proteins or by introduction of an M133T mutation, which generates an extra N-linked glycosylation site. The M133T mutation can coevolve with and rescue the virion secretion defect of the classic G145R immune escape mutation. Furthermore, through a collaboration with Dr. Lok, we will directly verify whether the immune escape mutants found in patient blood are enveloped virus particles, or primarily as nonenveloped core particles. Virion secretion is part of viral life cycle critical for viral infectivity and transmission to new hosts. Therefore, establishing possible secretion defect of the immune escape mutants and whether the defect can be overcome by co-infecting wild-type virus or secondary mutations may help predict the future threat posed by immune escape mutants and design better containment strategies. If virion secretion is restored by co-infecting wild-type virus or a compensatory second-site mutation, their infectivity in the presence of neutralizing antibodies can be tested in the near future in a cell line infectible with HBV. This study will provide important information for the prevention and treatment of HBV infection and associated liver cancer. PUBLIC HEALTH RELEVANCE: The vaccines to prevent hepatitis B virus infection sometimes fail because escape mutants arise. We would like to determine the efficiency of such mutants to release virus from infected cells and the impact of co-infecting wild-type virus or additional mutations. The knowledge obtained will help adjust the vaccine strategy for better control of these mutants.